A number of human diseases, including aplastic anemia and the myeloproliferative syndromes, are disorders of hematopoietic stem cells (HSC). The primary goal of this project is to develop a strategy for targeting gene expression to HSC in transgenic mice. This should provide a tool to study the molecular mechanisms important for normal and leukemic hematopoiesis. To accomplish this goal, we propose the following specific aims: (1) we will determine the genomic organization of the Ly-6 gene cluster in the region surrounding the mSca-1 locus, and (2) we will develop a system to target genes to the Sca-1+ compartment in mice using site-specific integration of a transgene, a via homologous recombination in embryonic stem (ES) cells. Sca-1 (Ly-6A/E) is a member of the Ly-6 family, a tightly clustered group of highly homologous genes localized to murine chromosome 15. We and others have noted that mutations made in transgenic mice that leave a selectable marker and its promoter in a targeted locus can result in unanticipated effects on the expression of other tightly linked genes. For this reason, we will characterize the murine Ly-6 locus in the region linked to Sca-1. Specific reagents will be generated to analyze the expression of genes "neighboring" the site of integration into the Sca-1 locus. Characterization of the murine cluster will be complemented by analysis of a syntenic region on human chromosome 8, recently found to contain several potential homologs of mLy-6 genes. Sca-1 "knock-in" mice derived from correctly targeted ES clones should coexpress the integrated transgene with the endogenous Sca-1 allele. Since essentially all long- term bone marrow repopulating activity in the strains of mice under analysis resides in the Sca-1+ compartment, this strategy should result in HSC targeting in vivo. A mutated hCD4 reporter gene will be utilized in an initial proof of principle experiment to establish correct targeting with this vector. Subsequent experiments will include targeting the oncoproteins bcl-2 and PML/RAR alpha to HSC in order to study the mechanism of leukemic transformation of this compartment. This work will be conducted under the supervision of Dr. Timothy Ley. The laboratory has considerable expertise in transgenic technology and in the analysis of murine hematopoiesis. An advisory committee consisting of internationally recognized experimental hematologists has been assembled. Core facilities of the Washington University Cancer Center in addition to the scientific and clinical resources of Washington University Medical School and the Barnes-Jewish Hospital (an 1100 bed tertiary care center) will provide an appropriate environment to facilitate this candidate's transition to independent research. The long-term goal of this investigator is to study the molecular bases of hematopoiesis and leukemogenesis as an active member of a clinical Hematology/Bone Marrow Transplant Division.